Modifying Rap1-signalling by targeting Pde6δ is neuroprotective in models of Alzheimer’s disease

Background

Neuronal Ca²⁺ dyshomeostasis and hyperactivity play a central role in Alzheimer’s disease pathology and progression. Amyloid-beta together with non-genetic risk-factors of Alzheimer’s disease contributes to increased Ca²⁺ influx and aberrant neuronal activity, which accelerates neurodegeneration in a feed-forward fashion. As such, identifying new targets and drugs to modulate excessive Ca²⁺ signalling and neuronal hyperactivity, without overly suppressing them, has promising therapeutic potential.

Methods

Here we show, using biochemical, electrophysiological, imaging, and behavioural tools, that pharmacological modulation of Rap1 signalling by inhibiting its interaction with Pde6δ normalises disease associated Ca²⁺ aberrations and neuronal activity, conferring neuroprotection in models of Alzheimer’s disease.

Results

The newly identified inhibitors of the Rap1-Pde6δ interaction counteract AD phenotypes, by reconfiguring Rap1 signalling underlying synaptic efficacy, Ca²⁺ influx, and neuronal repolarisation, without adverse effects in-cellulo or in-vivo. Thus, modulation of Rap1 by Pde6δ accommodates key mechanisms underlying neuronal activity, and therefore represents a promising new drug target for early or late intervention in neurodegenerative disorders.

Conclusion

Targeting the Pde6δ-Rap1 interaction has promising therapeutic potential for disorders characterised by neuronal hyperactivity, such as Alzheimer’s disease.

     

Alteration in mitochondrial Ca<Superscript>2+</Superscript> uptake disrupts insulin signaling in hypertrophic cardiomyocytes

Background

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca²⁺ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.

Results

In the present study we investigated insulin-dependent mitochondrial Ca²⁺ signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca²⁺-fluorescent probes we showed that insulin increases mitochondrial Ca²⁺ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca²⁺ uniporter, as well as by siRNA-dependent mitochondrial Ca²⁺ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca²⁺ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca²⁺ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.

Conclusions

Mitochondrial Ca²⁺ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

     

Short-term partitioning of Cd recently taken up between sunflowers organs (<Emphasis Type="Italic">Helianthus annuus</Emphasis>) at flowering and grain filling stages: effect of plant transpiration and allometry

Aims

This work concentrated on understanding the allocation of Cd recently taken up between the organs of sunflower at early and middle reproductive growth stages. The roles of transpiration and allometry were investigated.

Methods

Sunflowers were grown hydroponically in greenhouse, being exposed to low concentrations of Cd (pCd²⁺ = 11.03). At flower bud and grain filling stages, plants were exposed for three days to ¹¹¹Cd and at the same time, subjected or not to fans to increase the transpiration. The partitioning of ¹¹¹Cd between plant organs measured by high resolution ICP-MS was then modelled.

Results

Although the use of fans increased the plant water uptake and transpiration by about 20%, there were no significant effects on the partitioning of recent Cd. Most of the recent Cd was recovered in roots (60%) and only 2.8% were found in seeds (0.8% for the husk and 2.0% for the almonds). The sequestration of recent Cd in a plant organ was successfully explained by its biomass and except for leaves, by the biomass of other organs acting as competitive sinks.

Conclusions

This work proposes a modelling approach for the partitioning of the labelled Cd between plant organs in sunflower.

     

Airway smooth muscle relaxation results from a reduction in the frequency of Ca<Superscript>2+</Superscript> oscillations induced by a cAMP-mediated inhibition of the IP<Subscript>3</Subscript> receptor

Background

It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca²⁺ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca²⁺ oscillations.

Methods

The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca²⁺ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.

Results

All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca²⁺ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca²⁺ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca²⁺ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca²⁺ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca²⁺ available to stimulate contraction because an elevation of intracellular Ca²⁺ concentration induced by exposure to a Ca²⁺ ionophore (ionomycin) or by photolysis of caged-Ca²⁺ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca²⁺-activated K⁺ channels, which would be expected to increase Ca²⁺ influx and contraction. By contrast, the photolysis of caged-IP₃ in the presence of agonist, to further elevate the intracellular IP₃ concentration, reversed the slowing of the frequency of the Ca²⁺ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP₃R to IP₃ was reduced by FSK and this was supported by the reduced ability of IP₃ to release Ca²⁺ in SMCs in the presence of FSK.

Conclusion

These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca²⁺ oscillation frequency by reducing internal Ca²⁺ release through IP₃ receptors.

     

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

Background

A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive.

Findings

Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca²⁺ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca²⁺ in a concentration-dependent manner. Conversely, increasing Ca²⁺ levels inhibits kinase activity up to 500-fold at 100 nM Ca²⁺.

Conclusions

Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

     

Modelling the acid/base <Superscript>1</Superscript>H NMR chemical shift limits of metabolites in human urine

Introduction

Despite the use of buffering agents the ¹H NMR spectra of biofluid samples in metabolic profiling investigations typically suffer from extensive peak frequency shifting between spectra. These chemical shift changes are mainly due to differences in pH and divalent metal ion concentrations between the samples. This frequency shifting results in a correspondence problem: it can be hard to register the same peak as belonging to the same molecule across multiple samples. The problem is especially acute for urine, which can have a wide range of ionic concentrations between different samples.

Objectives

To investigate the acid, base and metal ion dependent ¹H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture.

Methods

Urine samples from five different individuals were collected and pooled, and pre-treated with Chelex-100 ion exchange resin. Urine samples were either treated with either HCl or NaOH, or were supplemented with various concentrations of CaCl₂, MgCl₂, NaCl or KCl, and their ¹H NMR spectra were acquired.

Results

Nonlinear fitting was used to derive acid dissociation constants and acid and base chemical shift limits for peaks from 33 identified metabolites. Peak pH titration curves for a further 65 unidentified peaks were also obtained for future reference. Furthermore, the peak variations induced by the main metal ions present in urine, Na⁺, K⁺, Ca²⁺ and Mg²⁺, were also measured.

Conclusion

These data will be a valuable resource for ¹H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for ¹H NMR spectra.

     

Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure

Background

One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.

Methods

We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.

Results

We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca²⁺ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.

Conclusion

Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca²⁺ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.

     

Root fungal colonisations of the understory grass <Emphasis Type="Italic">Deschampsia flexuosa</Emphasis> after top-canopy harvesting

Aims

Root fungal relationships in forest understory may be affected by tree harvesting. Deschampsia flexuosa forms a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi functioning in nutrient uptake, and a more loose association with dark septate endophytic (DSE) fungi. We asked how harvesting affects fungal colonisations and whether DSE is more prone to change than AM.

Methods

Deschampsia flexuosa plants were sampled close to a control or a cut tree after top-canopy harvesting in a primary successional site. Colonisations were studied using light microscopy. Shoot N%, vegetation cover and soil nutrients were determined.

Results

Tree harvesting did not affect vegetation and soil parameters, except potassium (K⁺) increasing near cut trees. AM colonisation did not change, while DSE increased. Shoot N% increased with increasing DSE near cut trees. Hyaline septate (HSE) hyphae and soil K⁺ and magnesium (Mg²⁺) were positively correlated near control trees. Lichen cover and HSE correlated negatively.

Conclusions

DSE colonisation increased but AM did not change after harvesting. Positive correlation of DSE with shoot N% near cut trees may suggest a role for DSE in favouring plant nitrogen uptake after disturbance in an open microsite. HSE may play a role in K⁺ and Mg²⁺ uptake.

     

Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in <Emphasis Type="Italic">Bacillus natto</Emphasis>

Objective

To study the effect of Ca²⁺ on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410.

Results

When the concentration of Ca²⁺ varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH₄Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K _m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl₂ (0.05–0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl₂ in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca²⁺ is an intracellular process.

Conclusion

Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

     

Effects of magnesium deficiency on magnesium uptake activity of rice root,evaluated using <Superscript>28</Superscript> Mg as a tracer

Aims

The mechanisms underlying magnesium (Mg) uptake by plant roots remain to be fully elucidated. In particular, there is little information about the effects of Mg deficiency on Mg uptake activity. A Mg uptake kinetic study is essential for better understanding the Mg uptake system.

Methods

We performed a Mg uptake tracer experiment in rice plants using ²⁸?Mg.

Results

Mg uptake was mediated by high- and low-affinity transport systems. The K _m value of the high-affinity transport system was approximately 70 μM under Mg-deficient conditions. The Mg uptake activity was promoted by Mg deficiency, which in turn fell to the basal level after 5- min of Mg resupply. The induced uptake rate was inhibited by ionophore treatment, suggesting that an energy-dependent uptake system is enhanced by Mg deficiency.

Conclusions

The Mg uptake changes rapidly with Mg conditions in rice, as revealed by a ²⁸?Mg tracer experiment. This technique is expected to be applicable for Mg uptake analyses, particularly in mutants or other lines.

     

Crop metabolomics: from diagnostics to assisted breeding

Background

Until recently, plant metabolomics have provided a deep understanding on the metabolic regulation in individual plants as experimental units. The application of these techniques to agricultural systems subjected to more complex interactions is a step towards the implementation of translational metabolomics in crop breeding.

Aim of Review

We present here a review paper discussing advances in the knowledge reached in the last years derived from the application of metabolomic techniques that evolved from biomarker discovery to improve crop yield and quality.

Key Scientific Concepts of Review

Translational metabolomics applied to crop breeding programs.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Key factors identified by proteomic analysis in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings’ response to long-term exposure to different phosphate levels

Background

Maize seedlings are constantly exposed to inorganic phosphate (Pi)-limited environments. To understand how maize cope with low Pi (LP) and high Pi (HP) conditions, physiological and global proteomic analysis of QXN233 genotype were performed under the long-term Pi starvation and supplementation.

Methods

We investigated the physiological response of QXN233 genotype to LP and HP conditions and detected the changes in ion fluxes by non-invasive micro-test technology and gene expression by quantitative real-time polymerase chain reaction. QXN233 was further assessed using vermiculite assay, and then proteins were isolated and identified by nano-liquid chromatography-mass spectrometry.

Results

A negative relationship was observed between Na⁺ and Pi, and Na⁺ efflux was enhanced under HP condition. Furthermore, a total of 681 and 1374 were identified in the leaves and roots, respectively, which were mostly involved in metabolism, ion transport, and stress response. Importantly, several key Pi transporters were identified for breeding potential. Several ion transporters demonstrated an elaborate interplay between Pi and other ions, together contributing to the growth of QXN233 seedlings.

Conclusion

The results from this study provide insights into the response of maize seedlings to long-term Pi exposure.

     

Mechanical stimuli modulate intracellular calcium oscillations: a pathological model without chemical cues

Objective

To control the oscillatory behavior of the intracellular calcium ([Ca²⁺]_i) concentration in endothelial cells via mechanical factors (i.e., various hydrostatic pressures) because [Ca²⁺]_i in these cells is affected by blood pressure.

Results

Quantitative analyses based on real-time imaging showed that [Ca²⁺]_i oscillation frequency and relative concentration increased significantly when 200 mm Hg pressure, mimicking hypertension, was applied for >10 min. Peak height and peak width decreased significantly at 200 mm Hg. These trends were more marked as the duration of the 200 mm Hg pressure was increased. However, no change was observed under normal blood pressure conditions 100 mm Hg.

Conclusion

We generated a simple in vitro model to study [Ca²⁺]_i behavior in relation to various pathologies and diseases by eliminating possible complicating effects induced by chemical cues.

     

A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

Background

There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation.

Results

We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca²⁺ channel (IP₃R).

Conclusions

These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation.

     

Non-invasive urinary metabolomic profiling discriminates prostate cancer from benign prostatic hyperplasia

Introduction

Prostate cancer (PCa) is one of the most common malignancies in men worldwide. Serum prostate specific antigen (PSA) level has been extensively used as a biomarker to detect PCa. However, PSA is not cancer-specific and various non-malignant conditions, including benign prostatic hyperplasia (BPH), can cause a rise in PSA blood levels, thus leading to many false positive results.

Objectives

In this study, we evaluated the potential of urinary metabolomic profiling for discriminating PCa from BPH.

Methods

Urine samples from 64 PCa patients and 51 individuals diagnosed with BPH were analysed using ¹H nuclear magnetic resonance (¹H-NMR). Comparative analysis of urinary metabolomic profiles was carried out using multivariate and univariate statistical approaches.

Results

The urine metabolomic profile of PCa patients is characterised by increased concentrations of branched-chain amino acids (BCAA), glutamate and pseudouridine, and decreased concentrations of glycine, dimethylglycine, fumarate and 4-imidazole-acetate compared with individuals diagnosed with BPH.

Conclusion

PCa patients have a specific urinary metabolomic profile. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be useful to discriminate PCa from BPH in a clinical context.

     

The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes

Background

Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies.

Results

Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X₇ receptor and the transient receptor potential channel, TRPV2, another Ca²⁺ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca²⁺ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.

Conclusions

The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca²⁺ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.

     

Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca<Superscript>2+</Superscript> sensitivity but not on Ca<Superscript>2+</Superscript> sparks or BK channels

Background

In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels leading to lowered SMC [Ca²⁺]_i and vasodilation. Here we investigated whether Ca²⁺ sparks regulate SMC global [Ca²⁺]_i and diameter in the spiral modiolar artery (SMA) by activating BK channels.

Methods

SMAs were isolated from adult female gerbils, loaded with the Ca²⁺-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca²⁺ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca²⁺ signals and vascular diameter were analyzed.

Results

Ca²⁺ sparks and waves were observed in pressurized SMAs. Inhibition of Ca²⁺ sparks with ryanodine increased global Ca²⁺ and constricted SMA at 40 cmH₂O but inhibition of Ca²⁺ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH₂O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH₂O was abolished at 60 cmH₂O, consistent with a greater Ca²⁺-sensitivity of constriction at 40 cmH₂O than at 60 cmH₂O. When the Ca²⁺-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH₂O.

Conclusions

The results suggest that Ca²⁺ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca²⁺-sensitivity of constriction.

     

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Introduction

Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.

Objectives

To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.

Methods

We present a double-labelling LC–MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.

Results

While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.

Conclusions

Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.

Graphical abstract

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